Our long term objective is to understand the differentiation of specialized cell types in the pancreas. Our general strategy is to study the beta-cell as a model to decipher the transcriptional network functioning in pancreatic progenitor cells and guiding their differentiation into specific mature cell types. Our Specific Aims for this funding period are directed toward understanding the role of a single factor, Nkx2.2, in this transcriptional network. Nkx2.2 plays a critical role in beta-cell differentiation and is uniquely expressed in early pancreatic progenitor cells, islet progenitor cells, and mature beta-cells, via distinct mechanisms in each cell type, making it a useful tool to understand how gene expression is differentially regulated in these related cell types. Our Aims are directed at two basic questions: Question 1: What distinguishes pancreatic progenitor cells at different stages of development and determines their ability to generate different cell types? Aim 1. Contrast gene expression between early and late pancreatic progenitor cells. We will sort the progenitor cells from different ages and compare their expression of transcriptional regulators. Candidate transcriptional regulators will be tested for their ability to regulate the Nkx2.2 1B promoter. The information generated will be used to understand how the gene expression program in pancreatic progenitor cells changes at different stages of pancreatic development. Question 2: How does Nkx2.2 regulate downstream genes? Aim 2. Test the role of Nkx2.2 in the translation of Nkx6.1. In the absence of Nkx2.2, Nkx6.1 protein levels in the fetal pancreas drastically decline after the secondary transition, but mRNA levels do not. We will test the role of the nkx6.1 5' and 3' UTR in this translational regulation by Nkx2.2, and the role of Nkx2.2 in the regulation of the Nkx6.1 IRES sequences. Aim 3. Determine how Rfx6 inversely regulates Nkx2.2 and Nkx6.1. We have recently found that Rfx6 plays a critical role islet cell differentiation in mice and humans, and that it activates Nkx2.2 expression and represses Nkx6.1 expression. We will determine how Rfx6 regulates these genes, and, in parallel with Aim 2, whether it opposes Nkx2.2 in regulating the expression of Nkx6.1 at the protein level. Aim 4. Determine how Nkx2.2 silences pHox2b expression. Islet Nkx2.2 and neural-crest pHox2B form a non-cell-autonomous negative feedback loop that regulates beta-cell proliferation. We will test the role of Nkx2.2-regulated islet signals in silencing pHox2B expression in pancreatic neural-crest cells. These studies will help explain how the differentiated state is established and maintained. Ultimately this information will help guide efforts to generate beta-cells for patients with diabetes.